Method for producing xylitol from lignocellulosic hydrolysates without detoxification

ABSTRACT

A method for producing xylitol by fermentation of lignocellulosic hydrolysates without detoxification is provided. By using the originally isolated yeast  Candida  sp., xylose can be effectively converted into xylitol. The invention also provides the  Candida  strain having high furfural tolerance, and is capable to produce xylitol from various types of non-detoxified lignocellulosic hydrolysates, in which the overall utilization of xylose in hydrolysate can reach over 95%.

CROSS REFERENCE TO RELATED PATENT APPLICATION

This application is a divisional of an U.S. application Ser. No.12/775,655, filed on May 7, 2010.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for producing xylitol fromlignocellulosic hydrolysates without detoxification, in which the xylosefermentation strain Candida sp. shows high furfural tolerance and isused to convert xylose into xylitol from various source ofnon-detoxified lignocellulosic hydrolysates. The overall utilization ofxylose in hydrolysate reach over 95%.

2. Related Art

Xylitol is a rare sugar that exists in low amount and is the constituentof many vegetables and fruits. Xylitol is also one of the intermediatemetabolites in the sugars metabolism of mammalian, and its chemicalproperty belongs to the pentitols. The reason why xylitol attractsglobal attention is that it is a natural sweetener having equivalentsweetness to sucrose with the calorie of merely 60% of sucrose. Medicalstudies have shown that, xylitol is helpful for the prevention of dentalcaries, because it is not easily utilized by Streptococcus mutans andother microorganisms that may cause tooth decay, and also has thefunction of maintaining acid-base balance in the mouth. Furthermore,studies also point out that xylitol is rapidly metabolized to generateenergy, but the metabolism in human body dose not need insulinparticipation, so it is widely used as substitute of sucrose in thenutrition for diabetics in clinic presently. Generally, the developmentof industrial production of xylitol has applications for at least threemajor industries, that is, food processing industry (for example, bakingindustry, emulsifiers, stabilizing agents, and chewing gum),odontological prevention and control (dental caries prevention,promotion of tooth re-mineralization and rehardening), andpharmaceutical industry (prevention of upper respiratory tractinfection, as sweetener for its high-sweetness and low-calorieproperties, nutraceuticals, and vitamin formulations).

Presently, the method of industrial mass production of xylitol includesthe following steps. The lignocellulosic biomass material enriched withhemicellulose is pretreated by acid hydrolysis and converted into ahydrolysate with xylose as the main component. Next, the xylose-richhydrolysate is hydrogenated at high temperature and high pressure withthe catalysis of nickel metal and then to produce xylitol from theconversion of xylose. The yield of xylitol produced by this chemicalsynthesis is about 40-50%, and at the same time, all the sugars presentin the rhydrolysate are also reduced to their corresponding sugaralcohols. Therefore, in addition to xylitol, other sugar alcohols, suchas arabitol and sorbitol, may also exist in the product. These sugaralcohols have similar chemical properties and are relatively difficultto be separated. Furthermore, the production process of chemicalsynthesis is complex and consumes a great amount of energy, and theequipment cost is high, so the price of xylitol cannot be decreased. Inorder to decrease the production cost and meet the increasing marketdemand for xylitol, the industry is actively developing a high yield butlow energy-consumption alternative for xylitol production.

The bioconversion method for xylitol production by fermentation oflignocellulosic hydrolysate using microorganisms is the mostadvantageous and competitive alternative presently, in which by usingthe naturally occurring xylose-fermenting microorganisms, the xylose isdirectly converted into xylitol through the physiological metabolism ofthe microorganisms, and then the xylitol product is then recovered bypurification and crystallization. In addition to high production yield,bioconversion method also has the advantage in the elimination of therisk that the xylitol product may be contaminated by heavy metal by thechemical synthesis method. As for the regulation standard for foodadditives, the xylitol product produced by bioconversion method isrelatively safe.

In the relevant literatures currently collected, the type of thehydrolysates discussed in the studies of converting xylose into xylitolby microorganisms include corncob, corn fiber, sugarcane bagasse,hardwood, eucalyptus, walnut shell, brewer's spent grain, prairie grass,wheat straw, and rice straw, etc., among which, there is a largedifference in relevant xylitol yield (0.2-0.8 g/g).

Generally, the lignocellulosic biomass material mainly contains 60-80%of cellulose, hemicellulose, and 15-25% of lignin, in whichhemicellulose is required to be converted into pentoses (mainly xylose)through a pretreatment process, and then further converted into xylitolby microorganism fermentation. Presently, the pretreatment technologyfor converting hemicellulose into saccharides mostly adoptshigh-temperature and high-pressure thermal chemical pretreatmenttechnologies, such as, dilute acid hydrolysis, dilute acid-catalyzedsteam explosion to decompose hemicellulose into xylose. During thereaction of such pretreatment technology, generally, a certainproportion of raw material and an aqueous solution are firstly filledinto a reactor, and then 1-3% (w/w) of dilute sulfuric acid is addedunder high-temperature and high-pressure reaction conditions, and theliquid obtained after the reaction is so-called as hydrolysate. Inaddition to the release of sugars, the pretreatment also generatescertain amounts of fermentation inhibitors, such as, acetic acid,furfural, and hydroxymethyl furfural, accompanied with differentreaction conditions. Therefore, presently, the xylose-rich hydrolysateobtained by pretreatment is usually treated with detoxificationtechnology, such as, overliming, active carbon adsorption, and ionexchange resin, alone or in combination, such that hydrolysate issubjected to fermentation with microorganisms successfully and convertedxylose into the xylitol. For example, for the mostly used overlimingmethod (as shown in FIGS. 1A and 1B), the conditioning of thexylose-rich hydrolysate includes heating, adding excessive lime,solid-liquid separation, and adding an acid agent to adjust the pH valueto be weakly acidic, etc. The generated gypsum sludge is required to befurther treated and disposed. Because during the conditioning of theoverliming method, xylose loss is generally caused, and calcium sulfatesludge is generated, additional cost and equipments for treatment anddisposal are required, thus increasing the production cost of xylitol.By comparison, the process without detoxification is simple, and it isonly required to add an alkali agent to adjust the pH value of thexylose hydrolysate to be weakly acidic. However, the non-detoxifiedhydrolysate may contain high concentration of fermentation inhibitors,such as furfural and sulfate ion, so that the difficulty of convertingxylose into xylitol by fermentation is increased relatively. Therefore,it is an important issue for reduce the xylose loss as well as theproduction cost for xylitol to improve the competitiveness ofbioconversion-based xylitol production.

SUMMARY OF THE INVENTION

Accordingly, in order to solve the problems as described above, thepresent invention provides a method for producing xylitol byfermentation of lignocellulosic hydrolysates without detoxification. Axylitol fermentation yeast strain is isolated and screened from thefermentation broth of a 100 liter pentose fermenter in the mini-pilotplant of the Institute of Nuclear Energy Research for cellulosic ethanolresearch and development, and then molecular analysis of the 18S rDNAsequence has identified the strain as a yeast strain of Candida sp.belonging to Candida genus. The strain is used to ferment the syntheticxylose solution to produce xylitol, and the maximum yield can beapproximately to 0.8 g/g, which is higher than those of xylitolproduction strains also belonging to Candida genus, such as Candidaboidinii, Candida guilliermondii, Candida utilis, and Candida maltosa(mostly with a xylitol yield of below 0.7 g/g). Meanwhile, the xylitolyield of this newly isolated strain also reaches a leading level,compared with other xylitol-producing stains belonging to differentyeast genus.

In addition, the strain of the present invention has high tolerance tothe toxic inhibitor furfural, which is often existed in the pretreatedlignocellulosic hydrolysate. The fermentation experiments with asynthetic xylose solution supplemented with furfural show that thestrain can grow in the xylose solution containing 3 g/L of furfural andproduce xylitol, and the yield is very similar to that of the xylosefermentation without furfural.

On the other hand, by using various types of pretreated lignocellulosichydrolysates, such as, rice straw, silvergrass, sugarcane bagasse,napiergrass, pineapple peel, it is confirmed in the present inventionthat the strain can indeed directly grow in these lignocellulosichydrolysates without detoxification, and can effectively convert xylosein the lignocellulosic hydrolysates into xylitol.

Further scope of applicability of the present invention will becomeapparent from the detailed description given hereinafter. However, itshould be understood that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description given herein below for illustration only, and thusare not limitative of the present invention, and wherein:

FIGS. 1A and 1B show steps of overliming conditioning and conditioningwithout detoxification for xylose-rich lignocellulosic hydrolysate;

FIG. 2 shows the xylitol yield from the fermentation of synthetic xylosesolution and rice straw hydrolysate without detoxification;

FIG. 3 shows the influence of furfural on the xylitol yield; and

FIG. 4 shows the generation of xylitol by fermentation of rice strawhydrolysate without detoxification.

DETAILED DESCRIPTION OF THE INVENTION

The features and implementation of the present invention are describedin detail with preferred embodiments below.

I Results of Xylitol Production by Fermentation of Xylose Solution withCandida sp.

The xylitol production from xylose fermentation by Candida sp. strain isinvestigated by a synthetic xylose solution (YPX, medium of yeastextract, peptone and xylose), and the production of xylitol by the yeaststrain under different initial xylose concentrations is performed in a250 mL flask containing 50 mL of fermentation medium. The fermentationparameters are controlled, such that the temperature is 30° C., theagitation of the fermentation is 100 rpm, and the ratio of the inoculasize of the culture and the volume of the fermentation medium is 1:6(v/v). The results are shown in FIG. 2. The initial xylose concentrationin the fermentation medium will determine the final yield for xylitolproduction. As the initial xylose concentration is increased, thexylitol yield of the strain obtained by the fermentation is increasedcorrespondingly in a linear trend. When the xylose concentration is in arange of 20-80 g/L, the yield of xylitol produced by the strain is about0.55-0.70 μg; when the xylose concentration is higher than 80 g/L, theyield of xylitol produced by the strain is up to 0.75 g/g. Therefore,the initial xylose concentration surely has influence on the xylitolyield.

II the Tolerance of the Strain Candida sp. to Furfural

The fermentation medium contained 97 g/L sterilized synthetic xylosesolution with furfural in a concentration range of 1-3 g/L added. Thefermentation temperature is controlled at 30° C., the agitation of theincubator is maintained at 150 rpm, and the ratio of the inocula size ofthe culture and the volume of the fermentation medium is 1:6 (v/v). Theresults are shown in FIG. 3. Even at a high concentration of furfural of3 g/L, the xylose fermentation capacity of the strain is the same asthat without furfural, and the xylitol yield for all could be higherthan 0.7 g/g, thereby, it is obvious that the tolerance of the strain tofurfural is higher than 3 g/L.

III Xylitol Production by Fermentation of Rice Straw Hydrolysate withoutDetoxification Using the Candida Sp. Strain

The rice straw xylose hydrolysate is obtained by a pretreatment facilityequipped with a twin-screw extruder and a washing reactor. In thereaction, the suitably sized rice straw is firstly structurallydecomposed by the twin-screw extruder, in which the dilute acidconcentration is 1-3% (w/w), the screw speed is 40 rpm, the reactiontemperature is 120-130° C., the reaction time is 10-20 min, and theratio of the dry weight of the feeding rice straw and the aqueoussolution is about 50:100. After being treated with the extruder, therice straw is introduced to the washing reactor, into which anappropriate amount of steam is applied, such that the ratio of the dryweight of the rice straw and the aqueous solution is decreased to about30:100, and at the same time, the reaction temperature is raised to 160°C., and at this temperature, the reactant is boiled for 20 min. Then,the rice straw and the aqueous solution after reaction are discharged,and separated in a solid-liquid separation equipment. The obtainedaqueous solution is the xylose-rich hydrolysate, and the maincomposition is as shown in Table 1. The xylose concentration in thehydrolysate is about 30-35 g/L, NaOH is added into the rice strawhydrolysate to adjust to pH 6.0, the fermentation temperature iscontrolled at 30° C., the agitation of the incubator is maintained at100-150 rpm, the ratio of the inocula size of the culture and the volumeof the fermentation medium is 1:6 (v/v). The results are shown in FIG.4. The strain can completely consume xylose in the hydrolysate withoutdetoxification to produce xylitol in a reasonable time. Further comparedto the synthetic xylose solution, at the same xylose content, thexylitol production yield of the strain by fermentation of non-detoxifiedrice straw hydrolysate is even higher compared to fermentation of thesynthetic xylose solution (compared with FIG. 2).

TABLE 1 Composition of rice straw xylose hydrolysate Composition ofhydrolysate Concentration(g/L) Glucose 6.0~6.5 Xylose 32.3~35.2Arabinose 5.4~5.8 Acetic acid 1.7~2.2 Furfural 0.9-1.1 HMF 0.1-0.3

IV Xylitol Production by Fermentation of Sugarcane Bagasse XyloseHydrolysate without Detoxification Using Candida sp. Strain

The fermentation medium is a sugarcane bagasse hydrolysate pretreated bydilute acid. For pretreatment, the concentration of the dilute acid is1-4%, the operation temperature is 130° C., and the reaction time is 15min at this temperature. In this case, NaOH is added to adjust the pHvalue of the hydrolysate to 6.0, the fermentation temperature iscontrolled at 30° C., the agitation of the incubator is maintained at100 rpm, and the ratio of the inocula size of the culture and the volumeof the fermentation medium is 1:6 (v/v). The content of xylose in thesugarcane bagasse hydrolysate is 19.8-25.8 g/L, the content of theinhibitor furfural is 0.14-0.52 g/L, the amount of xylitol produced is7.74-12.51 g/L, and the overall utilization of xylose is 97.6-99%, asshown in Table 2.

TABLE 2 Xylitol production by fermentation of sugarcane bagassehydrolysate without detoxification glucose xylose xylitol ethanolfurfural time (h) (g/L) (g/L) (g/L) (g/L) (g/L)  0 2.27 19.82 1.76 0.1417 10.84 3.79 3.70 25 5.84 6.03 3.89 41 0.48 7.74 4.49  0 3.52 25.791.76 0.52 17 18.99 2.51 3.86 25 13.79 5.17 4.08 41 3.63 9.54 4.45 492.22 10.77 4.87 65 1.39 12.51 4.98 73 0.26 10.90 4.61

V Xylitol Production by Fermentation of Silvergrass Xylose Hydrolysatewithout Detoxification Using Candida sp. Strain

The fermentation medium is a silvergrass hydrolysate pretreated bydilute acid. For pretreatment, the concentration of the dilute acid is1-4%, the operation temperature is 130° C., and the reaction time is 15min at this temperature. In this case, NaOH is added to adjust the pHvalue of the hydrolysate to be 6.0, the fermentation temperature iscontrolled at 30° C., the agitation of the incubator is maintained at100 rpm, and the ratio of the inocula size of the culture and the volumeof the fermentation medium is 1:6 (v/v). The content of xylose in thesilvergrass hydrolysate is 17.5-23.14 g/L, the content of the inhibitorfurfural is 0.17-0.67 g/L, the amount of xylitol produced is 5.59-6.13g/L, and the overall utilization of xylose is 97%, as shown in Table 3.

TABLE 3 Xylitol production by fermentation of silvergrass hydrolysatewithout detoxification glucose Xylose xylitol ethanol furfural time (h)(g/L) (g/L) (g/L) (g/L) (g/L)  0 1.80 17.45 1.79 0.17 22 2.48 5.59 3.2726 0.72 5.58 3.35 30 0.45 5.28 2.88  0 3.43 23.14 1.91 0.67 22 17.201.10 3.88 26 15.78 1.73 4.34 30 12.50 2.61 4.70 46 2.75 6.02 5.18 501.24 6.13 5.23 70 0.65 6.10 3.10 78 6.19 2.20

VI Xylitol Production by Fermentation of Pineapple Peel XyloseHydrolysate without Detoxification Using Candida sp. Strain

The fermentation medium is a pineapple peel hydrolysate pretreated bydilute acid. For pretreatment, the concentration of the dilute acid is2%, the operation temperature is 130° C., and the reaction time is 15min at this temperature. In this case, NaOH is added to adjust the pHvalue of the hydrolysate to be 6.0, the fermentation temperature iscontrolled at 30° C., the agitation of the incubator is maintained at100 rpm, and the ratio of the inocula size of the culture and the volumeof the fermentation medium is 1:6 (v/v). The content of xylose in thepineapple peel hydrolysate is 19.8 g/L, and after 65 hr of fermentation,the amount of xylitol produced is 7.5 g/L, and the overall utilizationof xylose is up to 97.4%, as shown in Table 4.

TABLE 4 Xylitol production by fermentation of pineapple peel hydrolysatewithout detoxification glucose xylose xylitol ethanol furfural time (h)(g/L) (g/L) (g/L) (g/L) (g/L)  0 3.19 19.84 2.00 17 17.91 2.51 3.00 2514.86 1.46 3.12 41  7.61 4.29 3.50 49  4.24 5.48 3.64 65  1.67 7.52 3.7273  0.95 6.20 3.38

VII Xylitol Production by Fermentation of Napiergrass Xylose Hydrolysatewithout Detoxification Using Candida sp. Strain

The fermentation medium is napiergrass hydrolysate obtained by apretreatment process of acid-catalyzed steam explosion. Pretreatmentconditions: the concentration of the dilute acid is 2%, the operationtemperature is 180° C., the reaction time at this high temperature is 5min, after which the reactor is immediately depressurized. The reactionmix is then separated by a solid-liquid separation equipment to get theliquid fraction as hydrolysate. In this case, NaOH is added into thenapiergrass hydrolysate to adjust to pH 6.0, the fermentationtemperature is controlled at 30° C., the agitation of the incubator ismaintained at 100 rpm, the ratio of the inocula size of the culture andthe volume of the fermentation medium is 1:6 (v/v). The content ofxylose in the napiergrass hydrolysate is 15.55 g/L, the content of theinhibitor furfural is 1.19 g/L, and after 40 hr of fermentation, theamount of xylitol produced is 5.11 g/L, and the overall utilization ofxylose is up to 95.2%, as shown in Table 5.

TABLE 5 Xylitol production by fermentation of napiergrass hydrolysatewithout detoxification glucose xylose xylitol ethanol furfural time (h)(g/L) (g/L) (g/L) (g/L) (g/L)  0 6.99 15.55 1.34 1.19 16 0.21 10.62 1.575.04 24  7.06 2.89 5.42 40  1.39 5.11 5.78 48  0.41 3.74 4.66 64

As the compositions of lignocellulosic materials as mentioned above arevery different from each other, among the compositions of the pretreatedhydrolysates, in addition to the furfural having inhibitive effect onthe strain, a trace amount of inhibitors of different kinds that havenot been detected may exist. It can be known from the above that, theCandida strain of the present invention is applicable in xylosefermentation of various lignocellulosic hydrolysates withoutdetoxification, and can achieve a utilization rate of xylose of higherthan 95% while maintaining a reasonable efficiency for xylitolproduction, which indicates that the Candida strain of the presentinvention surely has a certain degree of tolerance to the fermentationinhibitors generally existing in the lignocellulosic hydrolysates, andif applied in the xylose fermentation, it will facilitate thesimplification of the conditioning steps of the xylose-richhydrolysates, thereby reducing the facilities investments for of thehydrolysate conditioning.

Although the specific embodiments have been illustrated and describedabove, it will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention.Furthermore, the present invention is not limited to the particularforms, and covers all modifications and variations of this inventionprovided they fall within the scope of the following claims and theirequivalents.

In view of the above, in terms of its general combination and features,the present invention has no been found in similar products, and has notbeen disclosed before its filing date. It indeed meets the requirementsof a patent and we thus propose this application according to theprovisions of the patent law.

What is claimed is:
 1. A method for producing xylitol by fermentation oflignocellulosic hydrolysates without detoxification, wherein a strainwith the capacity of xylose fermentation is used to convert xylose intoxylitol in lignocellulosic hydrolysates without detoxification.
 2. Themethod according to claim 1, wherein a pretreatment equipment isselected from a group consisting of an acid-catalyzed steam explosionsystem.
 3. The method according to claim 1, wherein the xylosefermentation strain is identified as the yeast strain belonging to theCandida.
 4. The method according to claim 1, wherein the carbon sourceof the xylose fermentation strain is selected from the group consistingof glucose, xylose, and xylose hydrolysate of a lignocellulosic biomassmaterial.
 5. The method according to claim 1, wherein thelignocellulosic material is selected from the group consisting of ricestraw, sugarcane bagasse, silvergrass, napiergrass, pineapple peel,switchgrass, wood, bamboo, and other lignocellulosic biomass materials.6. The method according to claim 1, wherein an alkali agent ispreviously added into the non-detoxified lignocellulosic hydrolysates toadjust the pH value between 4.5 and 7.0.